Integrin Cytoplasmic Tail Research Articles

An integrin αvβ3–c-Src oncogenic unit promotes anchorage-independence and tumor progression

Integrins regulate adhesion-dependent growth, survival and invasion of tumor cells. In particular, expression of integrin αvβ3 is associated with progression of a variety of human tumors. Here, we reveal a novel adhesion-independent role for integrin αvβ3 in pancreatic cancer and other carcinomas. Specifically, αvβ3 expressed in carcinoma cells enhanced anchorage-independent tumor growth in vitro and increased lymph node metastases in vivo. This required recruitment of c-src to the β3 integrin cytoplasmic tail, leading to c-src activation, crk-associated substrate (CAS) phosphorylation and tumor cell survival that, surprisingly, was independent of cell adhesion or focal adhesion kinase (FAK) activation. Reduced expression of endogenous αvβ3 or c-src not only suppressed anchorage-independent growth, but also decreased metastasis in vivo, yet did not affect migration/invasion. These data define an unexpected role for an integrin as a mediator of anchorage-independence suggesting that an αvβ3/c-src signaling module may account for the aggressive behavior of αvβ3-expressing tumors in man.

The beta3 integrin cytoplasmic tail: protein scaffold and control freak.

Platelet integrin alphaIIbbeta3 plays an essential role in thrombus formation through interactions with adhesive ligands. Successful parenteral blockade of these interactions has validated alphaIIbbeta3 as a therapeutic target in cardiovascular medicine. However, oral alphaIIbbeta3 antagonists have not been successful and there is an unmet need for more effective anti-platelet drugs. Growing evidence points to the cytoplasmic tails of alphaIIb and beta3, and the beta3 tail in particular, as scaffolds for intracellular proteins that mediate inside-out signaling and regulate alphaIIbbeta3 affinity for ligands. Intracellular protein interactions with the integrin cytoplasmic tails also regulate outside-in signals to the actin cytoskeleton. Here we focus on recent studies that illustrate the relevance of the beta3 cytoplasmic tail as a regulatory scaffold in vivo. We speculate that this scaffold or its interacting proteins may serve as therapeutic targets for the development of future anti-thrombotic drugs.

Recruitment of vimentin to the cell surface by β3 integrin and plectin mediates adhesion strength

Much effort has been expended on analyzing how microfilament and microtubule cytoskeletons dictate the interaction of cells with matrix at adhesive sites called focal adhesions (FAs). However, vimentin intermediate filaments (IFs) also associate with the cell surface at FAs in endothelial cells. Here, we show that IF recruitment to FAs in endothelial cells requires beta3 integrin, plectin and the microtubule cytoskeleton, and is dependent on microtubule motors. In CHO cells, which lack beta3 integrin but contain vimentin, IFs appear to be collapsed around the nucleus, whereas in CHO cells expressing beta3 integrin (CHOwtbeta3), vimentin IFs extend to FAs at the cell periphery. This recruitment is regulated by tyrosine residues in the beta3 integrin cytoplasmic tail. Moreover, CHOwtbeta3 cells exhibit significantly greater adhesive strength than CHO or CHO cells expressing mutated beta3 integrin proteins. These differences require an intact vimentin network. Therefore, vimentin IF recruitment to the cell surface is tightly regulated and modulates the strength of adhesion of cells to their substrate.

Kindlin-1 and -2 Directly Bind the C-terminal Region of β Integrin Cytoplasmic Tails and Exert Integrin-specific Activation Effects

Integrin activation, the rapid conversion of integrin adhesion receptors from low to high affinity, occurs in response to intracellular signals that act on the short cytoplasmic tails of integrin beta subunits. Talin binding to integrin beta tails provides one key activation signal, but additional factors are likely to cooperate with talin to regulate integrin activation. The integrin beta tail-binding proteins kindlin-2 and kindlin-3 were recently identified as integrin co-activators. Here we report an analysis of kindlin-1 and kindlin-2 interactions with beta1 and beta3 integrin tails and describe the effect of kindlin expression on integrin activation. We demonstrate a direct interaction of kindlin-1 and -2 with recombinant integrin beta tails in pulldown binding assays. Our mutational analysis shows that the second conserved NXXY motif (Tyr(795)), a preceding threonine-containing region (Thr(788) and Thr(789)) of the integrin beta1A tail, and a conserved tryptophan in the F3 subdomain of the kindlin FERM domain (kindlin-1 Trp(612) and kindlin-2 Trp(615)) are required for direct kindlin-integrin interactions. Similar interactions were observed for integrin beta3 tails. Using fluorescence-activated cell sorting we further show that transient expression of kindlin-1 or -2 in Chinese hamster ovary cells inhibits the activation of endogenous alpha5beta1 or stably expressed alphaIIbbeta3 integrins. This inhibition is not dependent on direct kindlin-integrin interactions because mutant kindlins exhibiting impaired integrin binding activity effectively inhibit integrin activation. Consistent with previous reports, we find that when co-expressed with the talin head, kindlin-1 or -2 can activate alphaIIbbeta3. This effect is dependent on an intact integrin-binding site in kindlin. Notably however, even when co-expressed with activating levels of talin head, neither kindlin-1 or -2 can cooperate with talin to activate beta1 integrins; instead they strongly inhibit talin-mediated activation. We suggest that kindlins are adaptor proteins that regulate integrin activation, that kindlin expression levels determine their effects, and that kindlins may exert integrin-specific effects.

Talin mediates monocyte α4β1 integrin‐mediated migration, but not chemokine‐triggered α4β1 affinity up‐regulation or adhesion

α4β1 integrin plays an important role in monocyte adhesion and emigration. Activation by chemokines and chemoattractants is required to increase α4β1 integrin affinity and mediate adhesion to VCAM‐1 expressed by cytokine‐activated endothelium. We investigated the role of talin, a cytoskeletal protein that interacts with integrin cytoplasmic tails, in α4β1 integrin‐dependent functions using formyl peptide receptor‐transfected U937 monocytic cells. fMLP‐induced affinity up‐regulation, measured by binding of soluble LDV‐FITC peptide, was unaffected by siRNA knockdown of talin‐1 or sequestration of talin through expression of a dominant inhibitory talin‐binding fragment of phosphatidylinositol phosphate kinase type I‐90. Similarly, talin‐1 knockdown did not inhibit fMLP or SDF‐1‐stimulated adhesion to VCAM‐1. Talin was however, essential for PMA‐induced adhesion to VCAM‐1, but not PMA‐induced α4β1 integrin affinity up‐regulation. In addition, talin knockdown reduced fMLP‐driven, α4β1 integrin‐dependent chemotactic migration across VCAM‐1 coated filters.These results suggest that talin association with the cytoplasmic tail of α4β1 integrin is not involved in chemokine‐induced affinity changes or adhesion. However, talin plays an important role in α4β1 integrin‐mediated chemotactic migration and PMA‐induced adhesion to VCAM‐1, steps that follow affinity modulation. Funded by CIHR.

β1 Integrin Cytoplasmic Domain Residues Selectively Modulate Fibronectin Matrix Assembly and Cell Spreading through Talin and Akt-1

The integrin beta(1) cytoplasmic domain (tail) serves as a scaffold for numerous intracellular proteins. The mechanisms by which the tail coordinates these proteins to facilitate extracellular matrix assembly and cell spreading are not clear. This study demonstrates that the beta(1) cytoplasmic domain can regulate cell spreading on fibronectin and fibronectin matrix assembly through Akt- and talin-dependent mechanisms, respectively. To identify these mechanisms, we characterized GD25 cells expressing the beta(1) integrin cytoplasmic domain mutants W775A and R760A. Although cell spreading appears normal in R760A mutant-integrin cells compared with wild type, it is inhibited in W775A mutant cells. In contrast, both mutant cell lines show defective fibronectin matrix assembly. Inhibition of cell spreading, but not matrix assembly, in the W775A mutant cells is due to a specific defect in Akt-1 activation. In addition, we find that both W775A and R760A mutant integrins have reduced surface expression of the 9EG7 epitope that correlates with reduced recruitment of talin to beta(1) integrin cytoplasmic complexes. Down-regulation of talin with small interfering RNA or expression of green fluorescent protein-talin head domain inhibits matrix assembly in beta(1) wild-type cells, mimicking the defect seen with the W775A and R760A mutant cells. These results demonstrate distinct mechanisms by which integrins regulate cell spreading and matrix assembly through the beta(1) integrin cytoplasmic tail.

Migfilin, a Molecular Switch in Regulation of Integrin Activation

The linkage of heterodimeric (alpha/beta) integrin receptors with their extracellular matrix ligands and intracellular actin cytoskeleton is a fundamental step for controlling cell adhesion and migration. Binding of the actin-linking protein, talin, to integrin beta cytoplasmic tails (CTs) induces high affinity ligand binding (integrin activation), whereas binding of another actin-linking protein, filamin, to the integrin beta CTs negatively regulates this process by blocking the talin-integrin interaction. Here we show structurally that migfilin, a novel cytoskeletal adaptor highly enriched in the integrin adhesion sites, strongly interacts with the same region in filamin where integrin beta CTs bind. We further demonstrate that the migfilin interaction dissociates filamin from integrin and promotes the talin/integrin binding and integrin activation. Migfilin thus acts as a molecular switch to disconnect filamin from integrin for regulating integrin activation and dynamics of extracellular matrix-actin linkage.

NMR Solution Conformations and Interactions of Integrin αLβ2 Cytoplasmic Tails

The integrins are bi-directional signal transducers. Devoid of enzymatic activity, the integrin cytoplasmic tail serves as a hub for the recruitment of cytosolic proteins, and many of these are signaling molecules. The leukocyte-restricted integrin alphaLbeta2 is essential for the adhesion, migration, and proliferation of leukocytes. Here we report solution conformations and interactions of the alphaLbeta2 cytoplasmic tails by NMR analyses. The alphaL tail is characterized by three helical segments in the order of helix 1-3 that are connected by two loops with helix 3 having a number of nuclear Overhauser effect contacts with helix 1 and helix 2. The conformation of the beta2 tail is less defined with only a helical segment restricted at its N terminus. Acidic residues from the helix 2-loop-helix 3 motif of alphaL were found to be responsible for its binding to calcium ion. There were detectable interactions between alphaL and beta2 tails, involving helix 1 and helix 3 of the alphaL tail and the N-terminal helix of the beta2 tail. Talin head domain that contains the FERM domain showed binding affinity of Kd approximately 0.5 microm with the beta2 tail. The binding affinity of alphaL and beta2 tails is Kd approximately 2.63 microm. These data are in line with the activating property of talin head domain on alphaLbeta2 by which binding of talin head domain to beta2 tail disrupts the interface of the alphaL and beta2 tails that constrains alphaLbeta2 in a resting state.

Genetic perturbation of the putative cytoplasmic membrane-proximal salt bridge aberrantly activates α4 integrins

Abstractα4 integrins play a pivotal role in leukocyte migration and tissue-specific homing. The ability of integrins to bind ligand is dynamically regulated by activation-dependent conformational changes triggered in the cytoplasmic domain. An NMR solution structure defined a putative membrane-proximal salt bridge between the αIIbβ3 integrin cytoplasmic tails, which restrains integrins in their low-affinity state. However, the physiological importance of this salt bridge in α4 integrin regulation remains to be elucidated. To address this question, we disrupted the salt bridge in murine germ line by mutating the conserved cytoplasmic arginine RGFFKR in α4 integrins. In lymphocytes from knock-in mice (α4-R/AGFFKR), α4β1 and α4β7 integrins exhibited constitutively up-regulated ligand binding. However, transmigration of these cells across VCAM-1 and MAdCAM-1 substrates, or across endothelial monolayers, was reduced. Perturbed detachment of the tail appeared to cause the reduced cell migration of α4-R/AGFFKR lymphocytes. In vivo, α4-R/AGFFKR cells exhibited increased firm adhesion to Peyer patch venules but reduced homing to the gut. Our results demonstrate that the membrane-proximal salt bridge plays a critical role in supporting proper α4 integrin adhesive dynamics. Loss of this interaction destabilizes the nonadhesive conformation, and thereby perturbs the properly balanced cycles of adhesion and deadhesion required for efficient cell migration.

A Role for α-Actinin in Inside-out αIIbβ3 Signaling.

Integrin activation is regulated by many different biochemical signaling pathways through the integrin cytoplasmic tails. Multiprotein complexes assembled around the integrin cytoplasmic tail are linked to the actin cytoskeleton. Binding of the cytoskeletal proteins to integrin cytoplasmic tails leads to the conformational rearrangements of integrin extracellular domains that modulate their affinity. Talin-1 or Kindlin-3 has been identified as integrin activator complex proteins. α-Actinin also links the cytoplasmic domains of integrin β tails to actin filaments. We report here a new role for α-actinin in inside-out integrin activation. To explore the role of α-actinin in inside-out signaling, platelets were stimulated with protease-activated receptor (PAR) - activating peptides (AP) under non-stirring condition for up to 20 min. Immunoprecipitation with anti-αIIbβ3 followed by immnoblotting with anti-α-actinin revealed that in resting platelets α-actinin was constitutively associated with αIIbβ3. When platelets were stimulated by PAR1-AP, α-actinin was dissociated from αIIbβ3 as an initial step. Interestingly α-actinin re-bound to αIIbβ3 at 20 min after PAR1-AP stimulation. In contrast to PAR1-AP stimulation, the α-actinin dissociation from αIIbβ3 induced by PAR4-AP was long-lasting. To reveal the dynamic changes in αIIbβ3 activation, we recently developed initial velocity analysis for PAC1 binding. In brief, FITC-PAC1 was added to the activated platelets at indicated time points after stimulation and incubated for only 30 seconds to get the PAC1 binding velocity at the time points in question. The velocity of PAC1 binding reflects the relative numbers of activated αIIbβ3 at the time points. This initial velocity analysis more clearly revealed that PAR1-AP stimulation induced only transient αIIbβ3 activation, whereas PAR4-AP induced long-lasting αIIbβ3 activation. Moreover, the dissociation of α-actinin from αIIbβ3 appears to correlate with the time-dependent changes in the number of activated αIIbβ3. The kinetics of α-actinin-αIIbβ3 interaction was synchronized with tyrosine phosphorylation of α-actinin. When stimulated with PAR1-AP, α-actinin was de-phosphorylated rapidly and re-phosphorylated in late phase. PAR4-AP induced more prolonged de-phosphorylation of α-actinin than PAR1-AP. Thus, these results suggest that the interaction between α-actinin and αIIbβ3 may correlate with inside-out signaling induced by PAR1-AP and PAR4-AP. In platelets from a patient with Glanzmann thrombasthenia the phosphotyrosine profile of α-actinin was almost the same as that of control platelets in both PAR1-AP and PAR4-AP stimulation, confirming that these changes are not mediated αIIbβ3 outside-in signaling. In sharp contrast PAR4-AP stimulation failed to induce the sustained de-phosphorylation of α-actinin in P2Y12-ADP receptor deficient platelets. The blockade of P2Y12 with AR-C69931MX impaired the levels of activated αIIbβ3 induced by PAR4-AP, which correlated with the re-association of α-actinin. To further examine the role for α-actinin in integrin activation, α-actinin was overexpressed in human megakaryoblastic CMK cells and PAR1- AP induced PAC-1 binding to αIIbβ3 was assessed. Initial velocity analysis on CMK cells showed that overexpressed α-actinin inhibited PAR1-AP induced αIIbβ3 activation. These data imply that the binding of α-actinin to αIIbβ3 may regulate the levels of αIIbβ3 activation. Our observations may provide a new molecular framework for understanding the functions of β3 integrins in platelets.

Integrin Cross-talk in Endothelial Cells Is Regulated by Protein Kinase A and Protein Phosphatase 1

In endothelial cells (ECs) beta1 integrin function-blocking antibodies inhibit alphavbeta3 integrin-mediated adhesion to a recombinant alpha4-laminin fragment (ralpha4LN fragment). beta1 integrin sequestration of talin is not the mechanism by which beta1 integrin modulates alphavbeta3 integrin ligand binding. Rather, treatment of the ECs with beta1 integrin function-blocking antibodies enhances cAMP-dependent protein kinase (PKA) activity and increases beta3 integrin serine phosphorylation. The PKA inhibitor H-89 abrogates the effect of beta1 integrin function-blocking antibodies on beta3 integrin serine phosphorylation and EC-ralpha4LN fragment binding. beta3 integrin contains a serine residue at position 752. To confirm the importance of this residue in alphavbeta3 integrin-ralpha4LN fragment binding, we mutated it to alanine (beta3S752A) or aspartic acid (beta3S752D). Chinese hamster ovary (CHO) cells expressing wild type or beta3S752A integrin attach robustly to ligand. CHO cells expressing beta3S752D integrin do not. Because the beta3 cytoplasmic tail lacks a PKA consensus site, it is unlikely that PKA acts directly on beta3 integrin. Instead, we have tested an hypothesis that PKA regulates beta3 integrin serine phosphorylation indirectly through phosphorylation of inhibitor-1, which, when phosphorylated, inhibits protein phosphatase 1 (PP1). Treatment of ECs with beta1 integrin function-blocking antibodies significantly increases phosphorylation of inhibitor-1. Furthermore, blocking PP1 activity pharmacologically inhibits alphavbeta3-mediated cell adhesion to the ralpha4LN fragment when both PKA and beta1 integrin function are inhibited. Concomitantly, there is an increase in serine phosphorylation of the beta3 integrin cytoplasmic tail. These results indicate a novel mechanism by which beta1 integrin negatively modulates alphavbeta3 integrin-ligand binding via activation of PKA and inhibition of PP1 activity.

Functional analysis of the cytoplasmic domain of the integrin α1 subunit in endothelial cells

AbstractIntegrin α1β1, the major collagen type IV receptor, is expressed by endothelial cells and plays a role in both physiologic and pathologic angiogenesis. Because the molecular mechanisms whereby this collagen IV receptor mediates endothelial cell functions are poorly understood, truncation and point mutants of the integrin α1 subunit cytoplasmic tail (amino acids 1137-1151) were generated and expressed into α1-null endothelial cells. We show that α1-null endothelial cells expressing the α1 subunit, which lacks the entire cytoplasmic tail (mutant α1-1136) or expresses all the amino acids up to the highly conserved GFFKR motif (mutant α1-1143), have a similar phenotype to parental α1-null cells. Pro1144 and Leu1145 were shown to be necessary for α1β1-mediated endothelial cell proliferation; Lys1146 for adhesion, migration, and tubulogenesis and Lys1147 for tubulogenesis. Integrin α1β1–dependent endothelial cell proliferation is primarily mediated by ERK activation, whereas migration and tubulogenesis require both p38 MAPK and PI3K/Akt activation. Thus, distinct amino acids distal to the GFFKR motif of the α1 integrin cytoplasmic tail mediate activation of selective downstream signaling pathways and specific endothelial cell functions.

Urokinase-type Plasminogen Activator Receptor Induces Conformational Changes in the Integrin αMβ2 Headpiece and Reorientation of Its Transmembrane Domains

The glycosylphosphatidylinositol-linked urokinase-type plasminogen activator receptor (uPAR) interacts with the heterodimer cell adhesion molecules integrins to modulate cell adhesion and migration. Devoid of a cytoplasmic domain, uPAR triggers intracellular signaling via its associated molecules that contain cytoplasmic domains. Interestingly, uPAR changes the ectodomain conformation of one of its partner molecules, integrin alpha(5)beta(1), and elicits cytoplasmic signaling. The separation or reorientation of integrin transmembrane domains and cytoplasmic tails are required for integrin outside-in signaling. However, there is a lack of direct evidence showing these conformational changes of an integrin that interacts with uPAR. In this investigation we used reporter monoclonal antibodies and fluorescence resonance energy transfer analyses to show conformational changes in the alpha(M)beta(2) headpiece and reorientation of its transmembrane domains when alpha(M)beta(2) interacts with uPAR.

The Talin Rod IBS2 α-Helix Interacts with the β3 Integrin Cytoplasmic Tail Membrane-proximal Helix by Establishing Charge Complementary Salt Bridges

Talin establishes a major link between integrins and actin filaments and contains two distinct integrin binding sites: one, IBS1, located in the talin head domain and involved in integrin activation and a second, IBS2, that maps to helix 50 of the talin rod domain and is essential for linking integrin beta subunits to the cytoskeleton ( Moes, M., Rodius, S., Coleman, S. J., Monkley, S. J., Goormaghtigh, E., Tremuth, L., Kox, C., van der Holst, P. P., Critchley, D. R., and Kieffer, N. (2007) J. Biol. Chem. 282, 17280-17288 ). Through the combined approach of mutational analysis of the beta3 integrin cytoplasmic tail and the talin rod IBS2 site, SPR binding studies, as well as site-specific antibody inhibition experiments, we provide evidence that the integrin beta3-talin rod interaction relies on a helix-helix association between alpha-helix 50 of the talin rod domain and the membrane-proximal alpha-helix of the beta3 integrin cytoplasmic tail. Moreover, charge complementarity between the highly conserved talin rod IBS2 lysine residues and integrin beta3 glutamic acid residues is necessary for this interaction. Our results support a model in which talin IBS2 binds to the same face of the beta3 subunit cytoplasmic helix as the integrin alphaIIb cytoplasmic tail helix, suggesting that IBS2 can only interact with the beta3 subunit following integrin activation.

Integrin‐associated proteins as potential therapeutic targets

Integrins are adhesion receptors important for hematopoiesis, leukocyte trafficking, and formation of immunological synapses; hence, they may provide targets for therapeutic intervention in leukocyte-driven pathologies. Blocking integrin-ligand binding is one strategy for inhibiting integrins; however, a complete loss of integrin function can lead to mechanism-based toxicities. Because integrin alpha and beta subunits interact with a variety of other proteins to receive and transmit cellular signals, targeting these integrin-associated proteins may utilize alternative sites for intervention that lead to therapies with fewer side effects. This review summarizes integrin-associated proteins in leukocytes and focuses on four of these proteins with perceived therapeutic potential. Specific mutations in the alpha4 integrin cytoplasmic tail block or enforce binding to paxillin and thus modulate integrin signaling required for efficient cell migration. Similarly, the association of RAPL(NORE1B) with beta2 integrins may participate in adhesive and migratory events in leukocytes. The beta integrin cytoplasmic tail-binding protein talin is critical for increasing the affinity of integrins (activation), and blockade of talin binding can prevent leukocyte arrest on the endothelium. Finally, the membrane protein CD98 mediates beta1 and beta3 integrin signaling and may be involved in leukocyte functions. Identification of biologically important interactions of integrins and signaling proteins can thus pave the way to new strategies for manipulating leukocyte functions.

Cell Adhesion-dependent Cofilin Serine 3 Phosphorylation by the Integrin-linked Kinase·c-Src Complex

Integrin-linked kinase (ILK) is involved in signal transduction by integrin-mediated cell adhesion that leads to dynamic actin reorganization. Actin (de)polymerization is regulated by cofilin, the Ser(3) phosphorylation (pS(3)cofilin) of which inhibits its actin-severing activity. To determine how ILK regulates pS(3)cofilin, we examined the effects of ILK on pS(3)cofilin using normal RIE1 cells. Compared with suspended cells, fibronectin-adherent cells showed enhanced pS(3)cofilin, depending on ILK expression and c-Src activity. The ILK-mediated pS(3)cofilin in RIE1 cells did not involve Rho-associated kinase, LIM kinase, or testicular protein kinases, which are known to be upstream of cofilin. The kinase domain of ILK, including proline-rich regions, appeared to interact physically with the Src homology 3 domain of c-Src. In vitro kinase assay revealed that ILK immunoprecipitates phosphorylated the recombinant glutathione S-transferase-cofilin, which was abolished by c-Src inhibition. Interestingly, epidermal growth factor treatment abolished the ILK effects, indicating that the linkage from ILK to cofilin is biologically responsive to extracellular cues. Altogether, this study provides evidence for a new signaling connection from ILK to cofilin for dynamic actin polymerization during cell adhesion, depending on the activity of ILK-associated c-Src.

The N-terminal Domains of Talin Cooperate with the Phosphotyrosine Binding-like Domain to Activate β1 and β3 Integrins

The activation of integrin adhesion receptors from low to high affinity in response to intracellular cues controls cell adhesion and signaling. Binding of the cytoskeletal protein talin to the beta3 integrin cytoplasmic tail is required for beta3 activation, and the integrin-binding PTB-like F3 domain of talin is sufficient to activate beta3 integrins. Here we report that, whereas the conserved talin-integrin interaction is also required for beta1 activation, and talin F3 binds beta1 and beta3 integrins with comparable affinity, expression of the talin F3 domain is not sufficient to activate beta1 integrins. beta1 integrin activation could, however, be detected following expression of larger talin fragments that included the N-terminal and F1 domains, and mutagenesis indicates that these domains cooperate with talin F3 to mediate beta1 activation. This effect is not due to increased affinity for the integrin beta tail and we hypothesize that the N-terminal domains function by targeting or orienting talin in such a way as to optimize the interaction with the integrin tail. Analysis of beta3 integrin activation indicates that inclusion of the N-terminal and F1 domains also enhances F3-mediated beta3 activation. Our results therefore reveal a role for the N-terminal and F1 domains of talin during integrin activation and highlight differences in talin-mediated activation of beta1 and beta3 integrins.

Cooperative Role of the Membrane-proximal and -distal Residues of the Integrin β3 Cytoplasmic Domain in Regulation of Talin-mediated αIIbβ3 Activation

Integrin cytoplasmic tails regulate integrin activation that is required for high affinity binding with ligands. The interaction of the integrin beta subunit tail with a cytoplasmic protein, talin, largely contributes to integrin activation. Here we report the cooperative interaction of the beta3 membrane-proximal and -distal residues in regulation of talin-mediated alpha IIb beta3 activation. Because a chimeric integrin, alpha IIb beta3/beta1, in which the beta3 tail was replaced with the beta1 tail was constitutively active, we searched for the residues responsible for integrin activation among the residues that differed between the beta3 and beta1 tails. Single amino acid substitutions of Ile-719 and Glu-749 in the beta3 membrane-proximal and -distal regions, respectively, with the corresponding beta1 residues or alanine rendered alphaIIbbeta3 constitutively active. The I719M/E749S double mutant had the same ligand binding activity as alpha IIb beta3/beta1. These beta3 mutations also induced alphaVbeta3 activation. Conversely, substitution of Met-719 or Ser-749 in the beta1 tail with the corresponding beta3 tail residue (M719I or S749E) inhibited alpha IIb beta3/beta1 activation, and the M719I/S749E double mutant inhibited ligand binding to a level comparable with that of the wild-type alpha IIb beta3. Knock down of talin by short hairpin RNA inhibited the I719M- and E749S-induced alpha IIb beta3 activation. These results suggest that the beta3 membrane-proximal and -distal residues cooperatively regulate talin-mediated alpha IIb beta3 activation.

NPXY Motifs in the β1 Integrin Cytoplasmic Tail Are Required for Functional Reovirus Entry

Reovirus cell entry is mediated by attachment to cell surface carbohydrate and junctional adhesion molecule A (JAM-A) and internalization by beta1 integrin. The beta1 integrin cytoplasmic tail contains two NPXY motifs, which function in recruitment of adaptor proteins and clathrin for endocytosis and serve as sorting signals for internalized cargo. As reovirus infection requires disassembly in the endocytic compartment, we investigated the role of the beta1 integrin NPXY motifs in reovirus internalization. In comparison to wild-type cells (beta1+/+ cells), reovirus infectivity was significantly reduced in cells expressing mutant beta1 integrin in which the NPXY motifs were altered to NPXF (beta1+/+Y783F/Y795F cells). However, reovirus displayed equivalent binding and internalization levels following adsorption to beta1+/+ cells and beta1+/+Y783F/Y795F cells, suggesting that the NPXY motifs are essential for transport of reovirus within the endocytic pathway. Reovirus entry into beta1+/+ cells was blocked by chlorpromazine, an inhibitor of clathrin-mediated endocytosis, while entry into beta1+/+Y783F/Y795F cells was unaffected. Furthermore, virus was distributed to morphologically distinct endocytic organelles in beta1+/+ and beta1+/+Y783F/Y795F cells, providing further evidence that the beta1 integrin NPXY motifs mediate sorting of reovirus in the endocytic pathway. Thus, NPXY motifs in the beta1 integrin cytoplasmic tail are required for functional reovirus entry, which indicates a key role for these sequences in endocytosis of a pathogenic virus.

Characterization of calcium- and integrin-binding protein 1 (CIB1) knockout platelets: Potential compensation by CIB family members

Platelet aggregation requires activation of the alphaIIbbeta3 integrin, an event regulated by the integrin cytoplasmic tails. CIB1 binds to the cytoplasmic tail of the integrin alphaIIb subunit. Previous over-expression and knockdown studies in murine megakaryocytes demonstrated that CIB1 inhibits integrin alphaIIbbeta3 activation. Here we analyzed Cib1(-/-) mice to determine the function of CIB1 in platelets in vitro and in vivo. We found that although these mice had no overt platelet phenotype, mRNA level of CIB1 homolog CIB3 was increased in Cib1(-/-) megakaryocytes. In vitro binding experiments showed that recombinant CIB1, -2 and -3 bound specifically to an alphaIIb cytoplasmic tail peptide. Subsequent protein modeling experiments indicated that CIBs 1-3 each have a highly conserved hydrophobic binding pocket. Therefore, the potential exists for compensation for the loss of CIB1 by these CIB family members, thereby preventing pathologic thrombus formation in Cib1(-/-) mice.